We report the presence of species-specific lipoxygenase activity and oxylipin patterns, providing the first demonstration of enzymatic production of docosahexaenoic acid derivatives in marine diatoms. The differences observed in lipoxygenase pathways among the species investigated broadly reflected the relationships observed with phylogenetic markers, thus providing functional support to the taxonomic diversity of the individual species.Marine planktonic organisms, such as diatoms, are prospective sources of novel bioactive metabolites. Oxygenated derivatives of fatty acids, generally referred to as oxylipins, in diatoms comprise a highly diverse and complex family of secondary metabolites. These molecules have recently been implicated in several biological processes including intra- and inter-cellular signaling as well as in defense against biotic stressors and grazers. Here, we analyze the production and diversity of C Note-HEpETE) E, whereaIt is of note that the diatom extracts also contained minor products that did not give diagnostic fragmentation pattern in LC/MS/MS and that are not taken into consideration in this study .L. aporus (14-LOX), at C-15 of EPA and C-17 of DHA in L. danicus and L. hargravesii (15-LOX), at C-18 of EPA and C-20 of DHA in L. convexus (18-LOX) and, finally at C-5 in T. belgicus (5-LOX)  and proceeds with high stereospecificity ,31. As t (5-LOX) .16 fatty acids, hexadecatrienoic and hexadecatetraenoic acid [Leptocylindrus, thus providing the first finding of these compounds in marine organisms. In mammals, the oxylipins derived from peroxidation of DHA, i.e., the D-series resolvins (neuro-)protectin D1 and maresins, are important compounds with multiple activities as endogenous local mediators, with strong anti-inflammatory effects in addition to some immunoregulatory activities [As far as we know, only LOX derivatives of EPA and Coic acid ,25,32,33oic acid ,35. In ttivities ,37.L. danicus and L. hargravesii was established as S 18-HEPE and (±)20-HDoHE, respectively. As S enantiomers of HEPEs and HDoHEs are usually eluted later than the corresponding R isomers under the chromatographic conditions used for our analysis, these data suggest that LOX metabolism of L. convexus specifically produces 18S-HEPE and 20S-HDoHE. In L. aporus, the configuration of 14-HEPE remained unassigned due to the absence of reference compounds. However, this product gave a single chromatographic peak on chiral HPLC. All the hydroxyacids were almost optically pure (enantiomeric excess ~99%), which confirms the pivotal role of enzymatic oxidation in the biosynthesis of NVOs in diatoms.In order to corroborate the lipoxygenase specificity and complement the elucidation of the biosynthetic pathways, hydroxyeicosapentaenoic acids (HEPEs) and hydroxydocosahexaenoic acids (HDoHEs) of the different species of diatoms were purified on Reverse Phase-HPLC (RP-HPLC) and re-injected on chiral HPLC. The absolute configuration of each metabolite was determined by co-elution with racemic mixture and pure enantiomers. By these means, absolute stereochemistry of 15-HEPE and 17-HDoHE of hed as S . In L. cLeptocylindrus genus. Regardless of the positional specificity of the LOX pathways, hydroxyacids were always more abundant than the corresponding epoxy-alcohols, while levels of EPA derivatives were generally higher than the DHA metabolites  was comparable in L. danicus and L. hargravesii (35.5 ± 2.8 and 41 ± 3.2 µg µg−1 protein) but slightly lower in L. convexus (21.3 ± 1.7 µg µg−1 protein). Leptocylindrus aporus and T. belgicus showed the lowest levels among the species under investigation in this study.The absolute quantity of major oxylipins  showed no evident LOX activity at all, while the two strains of L. convexus differed for the presence of compound with m/z 353  were used in this study , sonicated for 1 min and centrifuged at 2000× g for 5 min. The supernatants were combined and extracted twice with equal volume of CH2Cl2. The organic layers were combined, dried over dry Na2SO4, then filtered and evaporated at reduced pressure. The dry extract was methylated with ethereal diazomethane for 30 min, and evaporated again under nitrogen flow.Culture pellets collected for oxylipin analysis were extracted and analyzed according to the methods described in literature . Briefly−1 and directly analyzed by LC-MS. The mass spectrometry (MS) method was based on a micro-Quadrupole time-of-flight  instrument equipped with an electrospray ionization (ESI) source in positive ion mode and a UV photodiode array  detector (scan range 205–400 nm) for a dual monitoring of the chromatographic runs. For ESI-QToF-MS/MS experiments, argon was used as collision gas at a pressure of 22 mbar. Chromatographic analysis was carried out on a reverse-phase high pressure liquid chromatography (RP-HPLC) using a linear MeOH/H2O gradient 75/25 to 100/0 in 30 min with a column flow of 1 mL min−1. One tenth of the column flow was channeled by a post-column split to the ESI+ (Q-Tof) MS analyzer and the remaining 9/10 to the UV PDA detector [Methylated extracts were dissolved in methanol to a final concentration of 1 µg µLdetector .Protein content was determined by the Lowry method , according to manufacturer’s instruction. Triton-X100 25% was utilized as cellular detergent. The standard curve was made using bovine serum albumin  and a UV/Vis spectrophotometer DU-730 . Quantification was achieved by interpolation from calibration curves at 655 nm.2O 70:30 to 80:20 for 30 min, followed by isocratic elution in 80:20, flow rate 1 mL min−1. Each purified compound was then analyzed by chiral phase HPLC using a Chiralcel OD-H   using hexane-isopropanol  at a flow rate of 1 mL min−1 [S)-HEPE, (±) 18-HEPE, (±) 17-HDoHE, 17(S)-HDoHE, (±) 20-HDoHE were purchased at Cayman Chemical Company .All methylated extracts (0.5 mg) were fractionated on reverse-phase high pressure liquid chromatography (RP-HPLC)  and the products of interest  were collected by UV and MS detection by using a split flow ratio of 1:300 (MS/UV) with a gradient from MeOH:HSmall subunit ribosomal DNA (SSU rDNA) sequences obtained in previous study were downloaded for the strains used in this study and aligned with Clustal-W. Phylogenetic analysis was done using PAUP, version 4.0b10 , and ML Leptocylindrus and Tenuicylindrus, are both characterized by oxylipin pathways requiring EPA as substrate. In analogy with previous reports on other diatoms, the enantiomeric excess of the corresponding HEPEs confirms the enzymatic control over the oxidative pathway with a preferential addition of oxygen to generate S enantiomers. In fact, the chemical data clearly indicate that L. danicus and L. hargravesii possess 15S-LOX enzyme, whereas T. belgicus is featured by a 5-LOX. Both activities are rather common in diatoms. On the other hand, L. convexus shows NVOs that are likely derived by 18S-LOX, while L. aporus is associated to a yet undetermined 14-LOX. As Leptocylindraceae constitute a basal clade in the diatom phylogeny, the EPA pathway might be ubiquitously present in marine diatoms unless the character has been lost over time. In addition, three out of four analyzed Leptocylindrus species, as an autapomorphic or as a symplesiomorphic character, produce comparatively high amounts of DHA and oxidize them into its derivatives. According to well-established model of LOX specificity [S-hydroperoxide of DHA by arachidonate-dependent 15-LOX in mammals [S-oxylipins of DHA are produced by 15S eicosapentanoate:LOX in L. danicus and L. hargravesii. To the best of our knowledge this is the first report of high levels of DHA enzymatic derivatives in diatoms and in marine organisms, although these compounds might have been overlooked so far because of their trace presence. As DHA derived oxylipins could reflect the DHA concentration in the diatom cells, further studies are necessary to address the distribution of these oxylipins in other diatom species.The two known Leptocylindraceae genera, cificity , the pre mammals ,47, it cLeptocylindraceae species and distribution of LOX activities and NVO patterns. Oxylipin-based discrimination of species indeed might provide a fertile interface to diatom morphological and molecular species identification, as well as a functional validation of the different taxa pointing at possible eco-physiological differences among these species.In the current study, we also report a good match between taxonomic classification of individual In this view, the finding of DHA derivatives adds new compounds to the known diatom oxylipins diversity, increasing the number of traits useful for the functional discrimination. Even for species-rich diatom genera, individual compounds or unique combinations of different compounds can reflect the species identity. On the other hand, the process of oxylipin synthesis is dynamic and is influenced by the intraspecific-genetic diversity and abiotic factors. Therefore, prior to its application as biomarker for functional diversity analysis, it is important to decipher the factors that trigger their synthesis and the physiological conditions that modulate the process. Thus, greater efforts in characterizing the pathways of oxylipin synthesis in diverse diatoms species and under different conditions not only can help understanding its applicability as a potential functional diversity biomarker, but can also shed light on numerous other processes of ecological relevance in which these molecules are involved.


Electrospun nanofibers have been widely used as substrata for mammalian cell culture owing to their structural similarity to natural extracellular matrices. Structurally consistent electrospun nanofibers can be produced with synthetic polymers but require chemical modification to graft cell-adhesive molecules to make the nanofibers functional. Development of a facile method of grafting functional molecules on the nanofibers will contribute to the production of diverse cell type-specific nanofiber substrata.Small molecules, peptides, and functionalized gold nanoparticles were successfully incorporated with polymethylglutarimide (PMGI) nanofibers through electrospinning. The PMGI nanofibers functionalized by the grafted AuNPs, which were labeled with cell-adhesive peptides, enhanced HeLa cell attachment and potentiated cardiomyocyte differentiation of human pluripotent stem cells.PMGI nanofibers can be functionalized simply by co-electrospinning with the grafting materials. In addition, grafting functionalized AuNPs enable high-density localization of the cell-adhesive peptides on the nanofiber. The results of the present study suggest that more cell type-specific synthetic substrata can be fabricated with molecule-doped nanofibers, in which diverse functional molecules are grafted alone or in combination with other molecules at different concentrations. Although the invention of electrospinning was disclosed in the 1930s, electrospinning-related studies have exponentially increased in the last few decades owing to the demands of nanofibrous structures for diverse applications ,3, whichPSCs attract a lot of attention for their potential to supply any kind of somatic cells in the body. In addition to embryonic stem cells (ESCs), which are mainly produced from a preimplantation embryo -26, the Herein, we examined the potentiating activity of a functionalized nanofiber substratum for cardiomyocyte differentiation in comparison with that of laminin-211. The PMGI nanofiber was selected to be functionalized through electrospinning owing to its proven rigidity that enables incorporation of small fluorescent molecules through co-electrospinning , similarw/v) PMGI polymer solution  as described previously [v/v) and mixed completely: concentrated stock solutions were 10 mM fluorescent molecules such as fluorescein and porphine dissolved in ethanol, 1 mM fluorescent peptides dissolved in dimethylsulfoxide (DMSO), and 10.7 nM peptide-labeled AuNPs dissolved in water. The mixed solution was loaded into a syringe equipped with a 21 one-fourth-gauge blunt-ended steel needle . To produce nanofibers, 8 kV was applied between the needle and a grounded collector, which was a silicon wafer covered by aluminum foil, separated 10 cm apart, while the PMGI solution was released continuously out of the syringe at a speed of 0.8 μL/min via a syringe pump. The diameters of the collected nanofibers ranged from 300 to 500 nm. Synthetic nanofiber substrata were fabricated by repeated pipeting of the collected nanofibers, which were fully soaked in autoclaved Milli-Q water , against the surface of the polystyrene dishes. Approximately, nanofibers produced with 4 μL of PMGI solution were used to coat a 35 mm dish. The unbound nanofibers were washed out with autoclaved Milli-Q water. The substratum was sterilized under UV light for 4 h before use.Electrospinning was processed with 13 % , was dissolved in DMSO at a 10-mM concentration and added to the 13 % PMGI solution at a 10 % (v/v) ratio. The fluorescent molecule-doped nanofibers were used to construct nanofiber substrata using 10-cm polystyrene dishes. Each substratum was incubated at 37°C in 10-mL phosphate-buffered saline (PBS). Every 5 d, the residual fluorescence intensities of the nanofiber substrata were measured using a fluorescent microscope  and the accompanying MetaMorph image analysis software . The size of the measured area, exposure time, and threshold were preset for the equal measurement of the fluorescence intensities of 5 different areas in each substratum. For the peptide-releasing test, 3 peptides were custom synthesized : a hydrophobic peptide composed of 6 leucines, a negatively charged peptide composed of 6 glutamic acids, and a positively charged peptide composed of 6 lysines. To monitor their release, a lysine labeled with fluorescein isothiocyanate (FITC) was added to each of the peptides. The FITC intensities in the nanofiber substrata were measured as described for the small fluorescent molecules, with which the amounts of the residual peptides in the nanofiber substrata were quantified.Each fluorescent molecule, the sodium salt of fluorescein (fluorescein) and the 5,10,15,20-Tetra(4-pyridyl)-21 Solutions of 20-nM AuNP were used to dope the nanofibers through electrospinning. The AuNP-doped nanofibers were fully soaked in Milli-Q water and fragmented by pipeting. For transmission electron microscopy (TEM) observation, microdroplets of the fragmented nanofibers or the AuNPs were deposited and dried on a plastic holey film covering a copper grid. TEM was done using a JEM-2200FS , operating at 200 kV. The fast Fourier transform (FFT) analysis was done with the DigitalMicrograph software package .t-butyl alcohol for 1 h. After washing with PBS, the cells were dried at 4 °C for 30 min. Then, the cells were covered with a 5-nm-thick gold layer through a sputtering at 200A for 15 sec. Samples were observed with a microscope . Using the same method, plain PMGI nanofibers were prepared for structural analysis with the SEM microscope.Mouse R1 ESCs were spread over unfixed PMGI nanofiber mesh and cultured for 1 week under growth medium, which was composed of DMEM-F12 supplemented with 15 % fetal bovine serum, 0.1 mM 2-mercaptoethanol, non-essential amino acids, and 1,000 U/ml mouse leukemia inhibitory factor  from Millipore . The cells were fixed using 1 % glutaraldehyde solution in PBS for 1 h, and then soaked in 100 % N-hydroxysuccinimide . To the AuNP solution being stirred at room temperature, we added 0.3-mM EDC and 0.75-mM NHS, stirred the solution for 45 min to activate the carboxyl group, and then filtered the solution 3 times with 25-mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer. A solution containing a 10-M excess of a mixture of cRGD peptide  and/or heparin-binding peptide I  was added dropwise to the AuNP solution, which was preadjusted to pH 7.5 with 100-mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, and stirred for 2 h. The AuNP solution was filtered 3 times using a 10,000-MWCO filter to remove the unreacted molecules and finally reconstituted with Milli-Q water. Each step of the labeling was confirmed by measuring the size of the AuNPs using a Zetasizer .The unconjugated AuNPs  were labeled with functional peptides using a 2-step method. First, a 1-mM mixture of 16-mercapto-hexadecanoic acid  and the polyethylene glycol (PEG)-based molecule, which was dissolved in ethanol at a 1:3 ratio (MHDA/PEG), was added to a basic AuNP solution  and stirred for 24 h. In most cases, the mole fraction of the thiols in the solution was similar to the mole fraction of the thiols bound to the nanoparticles. The solution was then filtered 3 times with a 10,000-MWCO filter  by adding Milli-Q water at each step. The second step involved linking the peptides to the AuNPs via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide  and Nanofiber meshes (1-mm thickness) were laid on a solid surface on which a water drop was placed. Static contact angles of the water drops were measured using the sessile drop method on a homemade contact angle instrument and the Low Bond Axisymmetric Drop Shape Analysis plugin  for Imag2) were seeded and incubated at 37°C in the presence of 5 % CO2 for 6 h. HeLa cell attachment was monitored using a microscope, and the bound cells were quantified by counting the detached cells after trypsinization from the 3 dishes of each nanofiber substratum.The nanofiber substrata were constructed with the PMGI nanofibers containing plain AuNPs, FLAG and PEG-labeled AuNPs, or cRGD and PEG-labeled AuNPs. Over the nanofiber substrata, the same number of HeLa cells (500 cells/cmA human ESC line (KhES-3) and a human iPSC line (IMR 90-1) were maintained on mitomycin C-treated mouse embryonic fibroblasts (MEFs) with primate ESC culture medium  containing 5 ng/mL basic fibroblast growth factor . The human ESC line was used in conformity with the guidelines for derivation and use of human embryonic stem cells of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan. Cardiac differentiation was carried out as described in a previous study , with miGenerally, molecule-doped nanofibers have been developed for controlled release of the doped molecules -41. In cSynthetic substrata for human PSCs is required to have a large amount of cell-adhesive molecules because of the low adhesion of human PSCs. To achieve this, we used a gold nanoparticle (AuNP) because its large surface area enables high-density labeling of functional molecules with low cytotoxicity ,43. In aNext, we investigated whether a peptide can be localized on the nanofiber surface using the PEG-labeled AuNP and electrospinning. The surface localization of the peptide was confirmed via molecular recognition by an antibody. To this end, AuNPs were labeled with the 3X FLAG peptide, which is widely used as a tagging sequence, through a MHDA linker, with or without PEG as depicted in the diagram  were spread over each substratum and incubated for 6 h. The cells grown on the nanofiber substratum containing cRGD revealed enhanced attachment and spreading, as determined by the formation of its unique shape indicated by the red arrows, whereas there was no cell or only a limited number of cells with such attachment and spreading on the substrata containing FLAG-AuNPs or PEG-AuNPs, respectively  on cell adhesion, respectively. HeLa cells (500 cells/cmP < 0.01). Unexpectedly, the mean colony number from the nanofiber substrata containing PEG-AuNPs was comparable with that from the laminin-coated substrata  and iPSCs (201B7), which were maintained over MEFs in the growth medium, became embryoid bodies (EBs) via suspension culture. The same number of the EBs were spread over 3 dishes of each substratum and then induced to differentiate into cardiomyocytes using the differentiation medium Figure A. After a Figure C. Thus, 2 nanofibers through electrospinning for the wavelength-dependent photoelectric response and for enhancing lithium-ion diffusion and charge transfer, respectively [Nanofibers are proven substrata for culturing somatic cells, but their application to PSCs has recently been investigated . Herein,ectively ,45. Unliectively . Therefoectively , and we ectively .The method described here has attractive points in terms of cost, convenience, and applicability: the PMGI nanofiber is cheap, and functionalization is achieved through simple co-electrospinning and can be applied to examine concentration-dependent effects and combinatorial effects of different functional molecules to the cells. The produced functionalized nanofibers can be applied to lithographic patterning of the nanofibers as described  for moreOur findings indicate that PMGI nanofibers can integrate small molecules, peptides, and functionalized AuNPs through electrospinning. Using this technique, we were able to fabricate synthetic nanofiber substrata that enhanced HeLa cell adhesion or potentiated cardiomyocyte differentiation of human PSCs.We believe that these results would serve as a foundation to fabricate diverse cell type-specific substrata.The authors declare that they have no competing interests.DJ, SP, JL MI, JB, QY, LL, and SK performed the research; DJ, YC, KL, and NN analyzed the data and wrote the manuscript. All the authors read and approved the final manuscript.Figure S1. SEM images of PMGI nanofibers. Nanofibers maintained their structure. (A) Structure of newly produced PMGI nanofibers. (B) A mouse ESC colony cultured over PMGI nanofibers for 1 week was observed.Click here for fileFigure S2. The surface of a polystyrene dish can be coated with PMGI nanofibers by pipeting. Rhodamine 6B  was premixed in 13 % PMGI solution and then incorporated into the nanofibers via electrospinning. A polystyrene dish (10 cm) was coated with the Rhodamine-doped nanofibers by pipeting the nanofibers repeatedly against the surface. The fluorescence image indicates that Rhodamine remains in the nanofiber under PBS, which was monitored with a fluorescence confocal microscope. Scale bars: 50 μm.Click here for fileFigure S3. TEM images of PEG-labeled AuNPs. High resolution TEM images showed formation of PEG layer on the surface of AuNPs. Red arrows indicate the PEG layer. Scale bars: A = 10 nm and B = 5 nm.Click here for fileFigure S4. TEM images of PMGI nanofiber containing higher number of AuNPs. Plain AuNPs (≅ 50 nM) were integrated into PMGI nanofiber through co-electrospinning with which a TEM image was taken. Scale bars: A = 0.5 μm and B = 100 nm.Click here for fileFigure S5. Contact angle measurements. The high-density PMGI nanofibers were fixed on a solid polydimethylsiloxane (PDMS) gel using adhesive silicon on which a drop of Mili-Q water was placed on the nanofiber meshes to measure its contact angle. Surface localization of the PEG-labeled AuNP made the nanofiber substratum hydrophilic, resulting in a decreased contact angle of the water drop. Au and Au-PEG indicate plain gold nanoparticle and polyethylene glycol-labeled gold nanoparticle, respectively.Click here for fileFigure S6. Labeling process of a AuNP with functional peptides. The surface of a 15-nm AuNP was labeled with 75 % PEG and 25 % MHDA using equal amounts of cRGD and HBP covalently conjugated through EDC/NHS coupling. EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, NHS: N-hydroxysuccinimide, and MHDA: 16-mercaptohexadecanoic acid.Click here for fileFigure S7. Size measurement of a functionalized AuNP.The size of an AuNP labeled with the 3X FLAG peptide (NH2-MDYKDHDGDYKDHDIDYKDDDDK-OH) through MHDA was measured. The surface of a 15-nm AuNP (A) was covered with 75 % PEG and 25 % MHDA (B), and the FLAG peptide was conjugated at the MHDA (C). The growing size of the AuNP after each labeling process was measured using a Zetasizer. The actual size of the functionalized FLAG-AuNP almost exactly matched the predicted size, which was calculated based on the length of the linker and 3X FLAG peptide. The results represent 3 independent measurements.Click here for file


This balance of phosphorylation control suggests that a D1 receptor agonist and a NMDA glutamate receptor antagonist should have additive or synergistic actions to increase activated DARPP-32 and consequent behavioral effects. This hypothesis was tested in a volitional consumption of ethanol model: the selectively bred Myers’ high ethanol preferring (mHEP) rat. A 3-day baseline period was followed by 3-days of twice daily injections of drug(s) or vehicle(s) and then a 3-day post-treatment period. Vehicle, the D1 agonist SKF 38393, the non-competitive NMDA receptor antagonist memantine, or their combination were injected 2 h before and after lights out. The combination of 5.0 mg/kg SKF 38393 with either 3.0 or 10 mg/kg memantine did not produce an additive or synergistic effect. For example, 5.0 mg/kg SKF reduced consumption of ethanol by 27.3% and 10 mg/kg memantine by 39.8%. When combined, consumption declined by 48.2% and the proportion of ethanol solution to total fluids consumed declined by 17%. However, the consumption of food also declined by 36.6%. The latter result indicates that this dose combination had a non-specific effect. The combination of SKF 38393 with (+)-MK-801, another NMDA receptor antagonist, also failed to show an additive effect. The lack of additivity and specificity suggests that the hypothesis may not be correct for this in vivo model. The interaction of these different receptor systems with intraneuronal signaling and behaviors needs to be studied further.Stimulation of the dopamine D1 receptor is reported to cause the phosphorylation of DARPP-32 at the thre34 position and activates the protein. If intracellular Ca A receptor, inhibition of excitatory glutamate (NMDA) receptors -decahydroisoquinoline-3-carboxylic acid), drugs that inhibit the binding of glycine to an accessory site on the receptor, (+)-HA-966 (3-amino-1-hydroxy-2-pyrrolidone) and ACPC (1-aminocyclo-propane-1-carboxylic acid), a drug that binds to the “PCP-site” to potently and non-competitively inhibit, dizolcipine (MK-801), or a drug that blocks the opened channel, memantine, all will decrease volitional consumption at doses with insignificant or small effects on food intake and motor performance  and either memantine or (+)MK-801, both non-competitive inhibitors of the glutamate NMDA receptor , memantine hydrochloride and (+)-MK-801 hydrogen maleate were purchased from Sigma-Aldrich . Drugs were dissolved in distilled water and doses were calculated as the free base. Saline was used as the vehicle control for all drugs.Table 1Effects of 2.5 mg/kg SKF-38393, 1.0 mg/kg memantine, or in combination b.i.d. for three days on daily ethanol consumption, proportion of fluids consumed and food intake.At the dose of 5.0 mg/kg s.c. SKF-38393 there occurred a significant decrease from the pre-treatment period in the consumption of ethanol by 42.8% and the proportion declined by 31.1% . Food inTable 2Effects of 5.0 mg/kg SKF-38393, 3.0 mg/kg memantine, or in combination b.i.d. for three days on daily ethanol consumption, proportion of fluids consumed and food intake.In Table 3Effects of 5.0 mg/kg SKF-38393, 10 mg/kg memantine, or in combination b.i.d. for three days on daily ethanol consumption, proportion of fluids consumed and food intake.1 receptors (25 µm SKF 38393) will increase production of cAMP, increase PKA activity, and activate DARPP-32. In turn, the NMDA receptor will become more phosphorylated and less sensitive to inactivation by ethanol [1 receptor or DARPP-32 in mice reduces ethanol-seeking behaviors [in vivo.Ethanol has the effect of causing the dopaminergic neurons in the mid-brain to discharge at increased rates and the effect is mediated through opiate receptors  and etha ethanol . Eliminaehaviors . In veryehaviors , a resulTable 4Effects of 2.5 mg/kg SKF-38393, 0.1 mg/kg (+)-MK-801 or in combination b.i.d. for three days on daily ethanol consumption, proportion of fluids consumed and food intake.1 receptor will reduce consumption of ethanol in a limited access paradigm [1 receptor agonist, SKF 38393, or a D1 receptor antagonist, SCH 23390, will reduce volitional consumption of ethanol by the mHEP rat [1 receptor reduce drinking suggests that deviation in either direction from normal dopaminergic function reduces the rewarding action of ethanol. SKF 38393 is a partial agonist at the D1 receptor and produces different behavioral effects as the dose is increased, but hyperactivity is not one [2 agonist [Stimulation of the Dparadigm  and duriparadigm . SystemimHEP rat . That bo not one . When us1 receptor agonist and an NMDA receptor antagonist should add or synergize to reduce the consumption of ethanol was based on the report by Nishi and co-workers [1 receptor agonist will activate DARPP-32 through the cAMP/PKA pathway and a NMDA agonist will counter the effect due to increased intracellular Ca2+ and activation of calcineurin. Their work suggests that a NMDA receptor antagonist would either further increase activated DARPP-32 or allow the enzyme to remain in the activated phosphorylated state. Doses of a D1 receptor agonist and NMDA antagonist were used that previously were demonstrated to decrease volitional consumption of ethanol with minimal reductions in food intake or locomotor disruption. Contrary to the hypothesis, there was neither an additive nor synergistic interaction when such drugs were combined. Neither memantine nor MK-801 combined with SKF 38393 caused a decrease of the consumption of ethanol than that seen with the D1 agonist alone. The hypothesis that a D-workers . A D1 re2+ will alter the activity of many enzymes in addition to calcineurin and the effects in a DARPP-32 expressing neuron will be different than in a neuron that does not.One explanation for the failure is that the shift in phosphorylation of DARPP-32 at Thre34 has no influence on the consumption of ethanol. It would be necessary to measure total and threo-34 phospho-DARPP-32 by western blot to determine whether changes in phosphorylation are occurring as predicted from in vitro studies. However, normally ethanol-seeking mice with DARPP-32 knocked out do not seek ethanol . The C57Another possible explanation is that memantine is too weak of an inhibitor of the NMDA glutamate receptor and perhaps is insufficient to reduce the calcium influx and activation of calcineurin (PP-2B). However, MK-801 is a more potent inhibitor of this receptor, but had no better effect when combined with SKF 38393. Or, it could be a matter of dosing. The doses of memantine and MK-801 were chosen based on prior work to have minimal motor and food consumatory effects. Large doses of these drugs produce an abnormal locomotor behavior, different from amphetamine-like stimulants  and in the case of MK-801 in extreme can produce a ketamine-like dissociative anesthetic state. The goal was to avoid these extreme effects and in order to demonstrate synergy, threshold effective doses should combine to produce a strong effect on consumption. Simply, this was not seen.in vivo situation other sources of calcium are available to activate calcineurin so that de-phosphorylation of DARPP-32 still occurs. That is, the amount of calcium entry associated with the NMDAR is small compared to other sources of calcium within the cell. The hypothesis was based on the results obtained in vitro by the application of drugs to striatal tissue slices [nucleus accumbens and medial pre-frontal cortex. More research needs to be done to determine the mechanism of interaction of NMDA receptor drugs with ethanol on dopaminergic activity and dopamine release.Finally, it may be possible that with the e slices  and may


Damage was also induced in WT mice by injection of cardiotoxin (CTX) into soleus muscle. Over-loading was applied for 14 days to the left soleus muscle in mdx and intact and CTX-injected WT mouse muscles by ablation of the distal tendons of plantaris and gastrocnemius muscles. All of the myonuclei in normal muscle of WT mice were distributed at the peripheral region. But, central myonuclei were noted in all fibers of WT mice regenerating from CTX-injection-related injury. Further, many fibers of mdx mice possessed central myonuclei and the distribution of such fibers was increased in response to over-loading, suggesting a shift of myonuclei from peripheral to central region. Approximately 1.4% branched fibers were seen in the intact muscle of mdx mice, although these fibers were not detected in WT mice. The percentage of these fibers in mdx, not in WT, mice was increased by over-loading (∼51.2%). The fiber CSA in normal WT mice was increased by over-loading (p<0.05), but not in mdx and CTX-injected WT mice. It was suggested that compensatory hypertrophy is induced in normal muscle fibers of WT mice following functional over-loading. But, it was also indicated that muscle fibers in mdx mice are susceptible to mechanical over-loading and fiber splitting and shift of myonuclei from peripheral to central region are induced.Effects of mechanical over-loading on the characteristics of regenerating or normal soleus muscle fibers were studied in dystrophin-deficient ( The muscles of mdx mice are characterized by cycles of muscle fiber necrosis followed by regeneration mdx muscle causes a reduction in force-generating capacity of muscle, and increases its sensitivity to mechanical stress-induced injury following the application of lengthening (eccentric) contraction mdx mice mdx mouse muscles The dystrophin-deficient  mouse muscles are not fully elucidated, either. Therefore, the current study was performed to test the hypotheses that responses of fiber properties to damage and/or over-loading are different between mdx and WT mice and that damaged fibers are fragile in response to the application of mechanical stress, not lengthening (eccentric) contraction However, it is still unclear how the functional over-loading affects the characteristics of mouse soleus muscle fibers. The responses of fiber properties to damage and regeneration in Guide for the Care and Use of Laboratory Animals of the Japanese Physiological Society. This study was also approved by the Committee on Animal Care and Use at Graduate School of Medicine, Osaka University (No. 22-071). All surgeries were performed under aseptic conditions with the mice under deep anesthesia .All experimental procedures were conducted in accordance with the mdx Jic (n = 5) and 4 and 5-week-old male C57BL/6J Jcl  mice were used. All mice were housed in a controlled environment with 12∶12 hr of light∶dark cycle and the temperature and humidity were maintained at ∼23°C and ∼55%, respectively. Two to three mice were housed in a cage with 11×20 cm and 11 cm height. Solid food  and water were supplied ad libitum.Five-week-old male C57BL/10-mdx mice (n = 5 each) were treated with 14 days of functional over-loading. Modified procedures, reported by Kawano et al.mdx and WT and CTX-injected WT mice (n = 5 each) under anesthesia by i.p. injection of sodium pentobarbital (5 mg/100 g body weight). The neural and vascular supplies were kept intact. The contralateral muscles were kept intact and served as the normal or CTX-injected control. The skin was sutured and the animals were allowed normal ambulation in the cage for 14 days. The behavior and daily activity of these mice in the cages were monitored by video filming during light period.In 4-week-old WT mice (n = 5), 5 µl of 0.3 mg/ml cardiotoxin  was injected into soleus muscles bilaterally to induce muscle injury. The CTX is known to damage the plasma membrane of myofibers, but leaves the basal lamina, satellite cells, and nerves intact allowing the rapid and reproducible muscle regeneration i.p. 2 days prior to the sampling. At the 14th day after surgery, the mice were anesthetized by i.p. injection of sodium pentobarbital (5 mg/100 g body weight). The soleus muscles were sampled bilaterally and cleaned of excess fat and connective tissue. For the longitudinal analyses of single fibers, the muscles sampled from mdx and WT mice with and/or without CTX-injection were stored in a freezing medium, named as “cellbanker”  at −80 °C.A single injection of 5-bromo-2′-deoxyuridine  in phosphate-buffered saline (PBS) was performed mdx Jic and C57BL/6J Jcl (WT) mice (n = 2 each) were also used additionally for analyses of cross-sectional and longitudinal photographic images of muscle fibers. Soleus muscles were sampled bilaterally. The right muscles were stretched equivalent to an in vivo length and pinned on a cork. They were frozen in isopentane cooled with liquid nitrogen. And the mid-portion of the frozen muscle was mounted on a cork perpendicularly by using optimum cutting temperature (OCT) compound , frozen in liquid nitrogen again, and stored at −80 °C for analyses of the location of myonuclei in the cross-sectional samples. The left muscles were stored in cellbanker  at −80 °C for three-dimensional analyses of myonuclear distribution.The control and similarly treated C57BL/10-The muscles stored in the cellbanker were thawed instantly at 35°C. The muscles were placed in Dulbecco's modified Eagle's medium  containing 0.06% type I collagenase, 1% antibiotics, and 10% new-born calf serum (NCS) for 3 hr at 35°C to digest the collagens. The collagenase-treated samples were fixed in 4% buffered-formaldehyde for 10 min and rinsed in PBS. Entire single muscle fibers were isolated from tendon-to-tendon using fine needles. The fibers were collected carefully using pipette to avoid any damage. The collected fibers were separated into three tubes  and immersed in DMEM containing 10% NCS.For immunohistochemistry, tube No. 1 and 2 were used to label M-cadherin and BrdU to measure the number of quiescent and mitotic active satellite cells, as described previously For the three-dimensional analyses of myonuclear distribution, the frozen left muscles, stored in the cellbanker, were also thawed and single muscle fibers were isolated as was stated above. Three-dimensional imaging of soleus muscle fibers was performed using a FV-300 confocal microscope system . The myonuclei were stained with PI (10 µg/ml PBS) for 5 min. The stained fibers were mounted on a slide glass with 50% glycerol in PBS. The fiber was scanned by an argon laser (488 nm of peak wave length) and computerized with a filter set for PI. Such scanning was repeated by moving the stage at 0.1 µm step width and the obtained images were saved as a multiple-TIFF file format. The image file was converted into a gray scale and three-dimensional image was constructed by merging all scanned pictures using 3D Viewer (Ratoc System Engineering).Cross-sections of the frozen right muscles, mounted on a cork, were cut (10-µm thickness) in a cryostat maintained at −20 °C. These sections were used for the analyses of the distribution of myonuclei. Staining of basal membrane was performed using the primary specific antibody against laminin . These sections were, then, blocked in 10% BSA diluted with PBS for 20 min at room temperature. The primary monoclonal antibodies specific for laminin, i.e., primary antibody, anti-laminin diluted at 1∶200 with PBS were applied to these sections, respectively. And the sections were kept at 4°C for at least 12 hr. The primary antibody was detected by secondary antibody reaction, which was performed for 2 hr at 4°C, using both goat anti-rabbit IgG conjugated to fluorescein  diluted at 1∶100 with PBS. After staining, these sections were rinsed with PBS. Immediately, the fibers on a slide glass were covered with a solution containing Hoechst 33342  to stain nuclei (myonuclei+satellite cells). A cover-glass, with “struts” of hardened nail polish on the corners to minimize fiber compression, was further placed over the samples. The stained images were analyzed by using a FV-300 confocal microscope with calibrated measurement software . The fibers with or without central nuclei were identified by visual examination.A FV-300 confocal microscope with an argon laser  was used to analyze the length and cross-sectional area (CSA) of fibers, number and CSA of the myonucleus, distribution of quiescent and mitotically active satellite cells, and number and location of NMJ. In the Hoechst-stained samples, the fibers with various distribution of myonuclei were classified into two types  as descrThe fibers in the tubes No. 1 and 2 were used to measure the fiber CSA and the number and CSA of the myonuclei. First, a maximum-intensity projection rotated orthogonally to the long axis of the fiber was produced from the stack, and the fiber CSA was measured at three non-overlapping regions randomly chosen along the fiber length. The Hoechst-labeled nuclei were counted in the entire single fiber. Myonuclear domain size Values are expressed as means ± SEM. Significant differences among groups were determined by using analysis of variance followed by Scheffé's post hoc test. Differences were considered significant at the 0.05 level of confidence.mdx mice were 60.3 and 39.7%, and type P fibers were further shifted to type C+P by over-loading . The data shown in Types of fibers were classified into two according to the distribution of myonuclei in peripheral (P) and/or central (C) region of single muscle fiber , and S2.mdx mice .Following the functional over-loading, the distribution of type P fibers decreased  and that of type C+P fibers with 0<∼20%, ∼40%, and ∼60% central nuclei insignificantly increased in mdx mice . And themdx mice was insignificantly decreased by 21.3% following functional over-loading, although that of type C+P fibers was insignificantly increased by 15.1%. The mean CSA of CTX-injected plus over-loaded type P fibers in WT mice was less than that of over-loaded normal muscles (p<0.05). The length of type C+P fibers of mdx mice without over-loading was shorter than those of CTX-injected WT mice . The response of the myonuclear number in mdx mice was identical between type P and C+P fibers. The number of myonuclei was also counted in the branched fibers.The myonuclear number per single fiber of both WT and -loading . The whoStatistically significant differences were not observed in the myonuclear domain size between any groups, although insignificantly greater value was noted in the over-loaded type P fibers of CTX-injected WT mice . This phmdx mice without application of mechanical load, although branched fibers were seen in type C+P fibers of mdx (1.4%) and CTX-injected WT mice (18.6%), respectively (mdx mice (10.5%) were not significant and the percentage of type C+P fibers of CTX-injected WT mice was stable. But approximately 51.2% increase of type C+P branched fibers was noted in the over-loaded mdx muscle (p<0.05).In general, no branched fibers were noted in the normal control and/or CTX-injected muscle of WT and ectively . The ovemdx mice, as well as the normal type C+P fibers of WT mice, disappeared in response to increased mechanical loading, even though those fibers were still existed (mdx mouse muscle (p>0.05). Quiescent satellite cells in type P fibers of CTX-injected WT and mdx mice, as well as the normal type C+P fibers of WT mice, were noted after over-loading .mdx and intact and CTX-injected WT mice to increase of mechanical loading were also studied. One of the hypotheses that responses of fiber properties to damage and/or over-loading are different between mdx and WT mice was accepted. Further, prominent fiber splitting in response to mechanical loading was noted in the muscle of mdx mice. But the muscle fibers of WT mice, even with damage, were resistive. Since the behavior and activity levels in the cage were similar between two kinds of mice with and without damage, such responses of fiber properties could be induced by functional over-loading, not daily activity level.It is thought that muscle fibers with central nuclei are at the emergence or regeneration stage generally mdx mice following functional over-loading. In CTX-injected WT mice, the myonuclei also tended to move from central to peripheral region in response to over-loading. These results indicated that muscle fiber degeneration in mdx mice was activated by over-loading. Such phenomena were also supported by the increased number of branched fibers. On the contrary, the functional over-loading tended to activate the recovery from the damage in CTX-injected WT mice. Approximately 14.9% of fibers shifted from type C+P to P (p>0.05).Shift of localization of myonuclei from peripheral to central region was induced in mdx and CTX-injected mice. Compensatory over-loading by ablation of synergists is known to result in increased muscle mass et al.mdx mice, although the CSA in type P fibers of normal WT mice increased, as was stated above. Such phenomena in the muscle of mdx mice may be related to the fiber splitting seen following over-loading.Hypertrophy of fibers was induced in response to mechanical over-loading in intact  WT mouse muscles, but not in mdx mice None of the characteristics of myonuclei, such as number, size, and domain, changed significantly by functional over-loading, although there was some insignificant trend in some parameters. Previously, we reported that these characteristics were identical regardless of the distribution of myonuclei in soleus muscle fiber of mdx mice in response to functional over-loading. It is known that branched or splitting muscle fibers could be found after muscle damage et al.The percent distribution of branched fibers was significantly increased in type C+P fibers of mdx mice. Exercise-induced damage mdx mice are fragile. It is interesting that such load-dependent splitting was not noted in the injured muscle of WT mice. The branched portion of these fibers had both peripheral and central nuclei. And the localization of NMJ in these fibers was similar to those of not-branched fibers. The data suggested that approximately 43.6% of type P fibers in mdx mice were shifted to type C+P fibers in response to over-loading. It is suggested that load-dependent damage or splitting may be induced mainly in type P fibers and the distribution of myonuclei may be shifted to central region of fiber accordingly.Increased splitting of fibers following mechanical over-loading, not by application of lengthening (eccentric) contraction et al.mdx mice and controls. However, it was also reported that satellite cells from mdx mice were more sensitive to fibroblast growth factor in culture than those from normal mice mdx mice was accelerated by the higher expression of myocyte enhancer factor 2 (MEF2) mdx mouse muscles may be always more active than those in WT mice. Therefore, we also counted the numbers of both mitotically active and quiescent satellite cells of the muscle fibers in the present study. However, no significant responses in the number of satellite cells were noted in response to over-loading, although the mean number of mitotically active cells in non-injured intact type P muscle fibers of mdx mice were greater than that of WT mice Click here for additional data file.Figure S2Three-dimensional structure of muscle fiber with myonuclei at both peripheral and central region.(MPG)Click here for additional data file.Figure S3Length and sarcomere number in soleus muscle fibers. Mean ± SEM. §: p<0.05 vs. CTX of WT in type C+P fibers. WT: wild type mice, mdx: dystrophin-deficient mice, CTX: cardiotoxin-injected, Norm: normal (without CTX injection), P and C+P: muscle fibers with myonuclear distribution at peripheral region only and both central and peripheral regions, respectively.(TIF)Click here for additional data file.Figure S4Myonuclear domain in WT, mdx, and CTX-injected WT mice. Mean ± SEM. See (TIF)Click here for additional data file.Figure S5Myonuclear cross-sectional area (CSA) in WT, mdx, and CTX-injected WT mice. Mean ± SEM. See (TIF)Click here for additional data file.Figure S6Number of satellite cells (SCs) in WT, mdx, and CTX-injected WT mice. Mitotically active satellite cells were not observed in the over-loaded type P muscle fibers in WT mice following CTX injection. Further, these cells were not seen in the over-loaded type P fibers of mdx mice and type C+P fibers of WT mice, either. Mean ± SEM. See (TIF)Click here for additional data file.


P value = 1.33E − 11) association of an SNP in the NCK2 gene on chromosome 2 with opiates addiction in African-origin men. Codependence analysis also identified a genome-wide significant association between NCK2 and comorbidity of substance dependence  in African-origin men. Furthermore, we observed that the association between the NCK2 gene  and opiates addiction reached the gene-based genome-wide significant level. In summary, our findings provided the first evidence for the involvement of NCK2 in the susceptibility to opiates addiction and further revealed the racial and gender specificities of its impact.Substance dependence is a complex environmental and genetic disorder with significant social and medical concerns. Understanding the etiology of substance dependence is imperative to the development of effective treatment and prevention strategies. To this end, substantial effort has been made to identify genes underlying substance dependence, and in recent years, genome-wide association studies (GWASs) have led to discoveries of numerous genetic variants for complex diseases including substance dependence. Most of the GWAS discoveries were only based on single nucleotide polymorphisms (SNPs) and a single dichotomized outcome. By employing both SNP- and gene-based methods of analysis, we identified a strong (odds ratio = 13.87) and significant ( Substance dependence is believed to result from a combination of genetic and environmental factors. Since substance dependence is a chronic brain disease, with high relapse rates, it causes serious social, economic, and medical consequences –3. The WLinkage and genome-wide association studies (GWASs) have implicated many regions and genes for dependence on alcohol, tobacco, and opiates. GABRA2, CHRM2, ADH4, PKNOX2, GABRG3, TAS2R16, SNCA, OPRK1, and PDYN have all been associated with alcohol dependence with various degrees of replication . AssociaPKNOX2 is associated with drug addiction in European-origin women. These examples underscore the necessity to consider demographic or even other covariates in genetic association studies.Complex diseases may involve heterogeneous genetic effects in different ethnic and gender groups , 44–47. NCK2 gene on chromosome 2 with opiates dependence in African-origin men at both the SNP and gene levels. NCK2 is a member of NCK family of adaptor proteins, which is associated with tyrosine-phosphorylated growth factor receptors of their cellular substrates [NCK2 has not been reported to be associated with any drug addiction outcomes in humans. Many of the reported genetic variants have been identified through single SNP association tests. Despite many of the successes, a single SNP tends to have a small effect, and the single SNP-based association tests require a very stringent significance level, which is likely a key factor to the so-called “missing heritability” problem , 49. To bstrates . Howeverhttp://www.ncbi.nlm.nih.gov/gap). SAGE is a large case-control association study which investigates the genetic variants for drug addiction. The samples were collected from three large-scale genome-wide association studies: Collaborative Study on the Genetic of Alcoholism (COGA), the Family Study of Cocaine Dependence (FSCD), and the Collaborative Genetic Study of Nicotine Dependence (COGEND) [P value for the Hardy-Weinberg equilibrium was set up by >0.0001. These steps reduced the level of noise in genotypes and increased the efficiency of analysis. There are 60 duplicate genotype samples and 9 individuals with ethnic backgrounds other than African origin or European origin. All of those individuals were removed from the subject list. Finally, there were a total of 3,627 unrelated samples with 859,185 autosomal SNPs for our final analysis. To alleviate the confounding by population substructure, we stratified the sample by race and sex. Finally, there are four sub-samples: 1,393 European-origin women, 1,131 European-origin men, 568 African-origin women and 535 African-origin men. The distribution of subjects diagnosed with lifetime dependence on substances in each of the six categories: nicotine, alcohol, marijuana, cocaine, opiates, or other drugs are presented in Phenotypes for multisubstance dependency and genome-wide SNP data from SAGE  were dow(COGEND) , 55, 56.(COGEND) , 57. IndP values for individual SNPs, and PLINK software (version 1.07) was used for analysis [r2 > 0.2) is calculated for the gene-based association test. The SNP-based association is performed by the standard allelic test and logistic regression to obtain the analysis . Meanwhime of independent P value among m SNPs within a gene. Then, we sort the SNPs and calculate the effective number me(j) of independent P-values among the top j significant SNPs. Finally, the modified Simes test [P value as follows,Pj)—based on the SNP association test results and LD files produced by PLINK. The procedure was performed as the following. We first calculate the effective number mes test  was empladers to  for detar2 > 0.9) with the initially mapped SNPs within the gene [In the gene-based method, SNPs within 20 kilo bases (kb) 5′ upstream and 10 kilo bases (kb) 3′ downstream of a gene's coding regions  were assthe gene . −6 as the genome-wide significance threshold for the gene-based association test. In contrast, we used 5.0 × 10−8 as the genome-wide significance threshold for the SNP-based association test [Since there are about 20,000 protein coding genes in human genome, we used 0.05/20, 000 = 2.5 × 10ion test . Although logistic regression is commonly used to study a binary outcome, it is not suitable to evaluate comorbidity involving multiple outcomes. We use a nonparametric association test based on Kendall's tau  to studyp-dimensional vector of traits Yi = (Yi(1),…, Yip)T, genotype Gi, and a q-dimensional vector of covariates Zi = (Zi(1),…, Ziq)T for the ith subject in a population-based study with n subjects, and { : i = 1,…, n} are independent samples. For subjects i and j, let Yi and Yj be their vectors of traits, respectively, and analogously, Gi and Gj and Zi and Zj are their genotypes and covariates. Generalized from Kendall's tau, a U statistic is defined to measure the association between Y and G as follows:U follows an asymptotically normal distribution in the absence of association [U statistic has been developed [Suppose that we observe a ociation . To accoeveloped , 65. We eveloped  for a deP < 1.0 × 10−4) in gene NCK2 on chromosome 2 (2q12) for opiates dependence in African-origin men. We identified a genome-wide significant SNP (rs2377339 with P = 1.33 × 10−11) for the opiates dependence in African-origin men by the allelic test. Logistic regression also yielded strong evidence for the association between the SNP rs2377339 (P = 1.01 × 10−7) and opiates dependence although the P-value did not reach the genome-wide significance threshold. In addition, P values from the gene-NCK2-based tests that were obtained through the standard allelic test and logistic regression are 3.12 × 10−10 and 2.70 × 10−6, respectively. The gene-based P value from the standard allelic test reached the genome-wide significance at gene level. The gene-based P value through logistic regression is very close to the gene-based genome-wide significance level. Therefore, both methods provided significant evidence that supports the association between the NCK2 gene and opiates dependence in African-origin men. For the addiction of the other five substances in African-origin men, nicotine dependence had the most significant association with the NCK2 gene (P = 9.56 × 10−3).The gene-based association results are displayed in the last two rows of NCK2 region. P value of 1.66 × 10−11. The genome-wide significant association between this haplotype and opiate addiction reduces the chance of a false discovery at the peak of a single SNP.We also examined association of haplotypes with opiate addiction in  We further examined the relationship between SNP rs2377339 and the opiates dependence in African-origin men. P values vary between different races and genders. The association between rs2377339 and opiates dependence becomes less significant in the overall cohort, after we adjusted race and gender in logistic regression.Furthermore, in NCK2 and comorbidity of substance dependence. The most significant signal in NCK2 was observed for SNP rs2377339 in men of African-origin with P = 3.65 × 10−8 in adjusted association test and P = 2.03 × 10−8 in unadjusted association test. P values of SNPs in NCK2 for other ethnicity by gender groups were far from the genome-wide significance level and, hence, are omitted here.In NCK2 gene that contains SNP rs2377339 also achieved the genome-wide significance for opiates dependence at the gene level. For the addiction of the other five substances, nicotine dependence had the most significant association but not significant at the genome-wide level. We found a genome-wide significant association between SNP rs2377339 and opiates dependence in African-origin men. The NCK2, a member of NCK family of adaptor proteins, is reported to be associated with tyrosine-phosphorylated growth factor receptors of their cellular substrate [NCK2 and nicotine dependence has been suggested in humans [NCK2 and drug addiction. ubstrate . The assn humans , 68. OurImportantly, about one-fifth of opiates addiction subjects in the African-origin men carried minor allele G of SNP rs2377339, which is more than 10-fold of the frequency in the nonopiates dependence group. This suggested that the minor allele G in SNP rs2377339 potentially elevates the risk for opiates dependence in African-origin men. We acknowledge that our analysis included only 44 African-origin men with opiates dependence. Therefore, it is important and necessary to validate our finding through independent and larger cohort studies. Specifically, there are two possible strategies to validate our finding. The direct approach is to replicate the association between SNP rs2377339 and opiates dependence in a larger cohort. An indirect approach is to evaluate whether SNP rs2377339 is associated with any substance dependence  as presented in NCK2 is significantly associated with substance dependence in African-origin men.A distinction of our analysis is to consider simultaneously multiple substance addictions rather than a single substance. This approach, which is a realistic depiction of substance dependence, confirmed that a novel susceptibility gene, This study has several limitations. First, we stratified by ethnicity and sex, which reduced sample sizes and affected the power of our analysis. Nonetheless, the significant associations revealed in African-origin men are consistent with the notion that men may be socially more prone to environmental influences that promote substance use and thus more vulnerable to addiction . Second,


Since its introduction to the synthetic community in 1984, Garner’s aldehyde has gained substantial attention as a chiral intermediate for the synthesis of numerous amino alcohol derivatives. This review presents some of the most successful carbon chain elongation reactions, namely carbonyl alkylations and olefinations. The literature is reviewed with particular attention on understanding how to avoid the deleterious epimerization of the existing stereocenter in Garner’s aldehyde. The universe is a dissymmetrical whole. I am inclined to think that life, as manifested to us, must be a function of the dissymmetry of the universe and of the consequences it produces. The universe is dissymmetrical; for, if the whole of the bodies which compose the solar system were placed before a glass moving with their individual movements, the image in the glass could not be superimposed on reality. Even the movement of solar life is dissymmetrical. A luminous ray never strikes in a straight line the leaf where vegetable life creates organic matter [...] Life is dominated by dissymmetrical actions. I can even foresee that all living species are primordially, in their structure, in their external forms, functions of cosmic dissymmetry.“ .–25.4 wit–95% ee) . AnotherS)-1 by Garner there have been many modifications and improvements to the synthesis of the enantiomers ((R)-1 and (S)-1). Modifications to the original synthesis have focused either on the reaction sequence (esterification first and then Boc protection) or on the reduction to aldehyde. Other groups have tried to improve the synthesis of the aldehyde. McKillop et al. found that the esterification reaction is performed best first with 235 mol % of HCl (formed in situ from the reaction of AcCl with MeOH) in MeOH . Wi. Wi4 . Th. Th1 cou7 and 9), peptide antibiotics (8), sphingosines and their derivatives /(R)-1, a new C–C bond is formed, hence allowing carbon chain elongation and further functionalization. Nucleophilic addition of an alkyne to 1 gives access to propargylic alcohols of the structure A (cis- (B) or trans-allylic alcohol C. cis-Selective reduction of A can be achieved with Lindlar’s catalyst with H2 under atmospheric pressure. The thermodynamic trans-allylic alcohol C arises from the reaction of A with Red-Al. Both isomers B and C can also be directly accessed from 1 with the corresponding cis- and trans-vinyl nucleophiles. Allylic alcohol B can be used as an intermediate in the synthesis of various natural products or intermediates thereof. The cis-double bond allows cyclizations to five- or six-membered rings. Five-membered dihydrofuran rings lead to the synthesis of furanomycin D . Th. ThS)-1 anti-products via the non-chelation pathway . Th. Th65]. S)-1 (27 and 28 in a 7:3 ratio (anti/syn). Addition of aggregation braking HMPT slightly improved the selectivity (10:3). Almost complete selectivity was achieved when BF3·Et2O (6 equiv) and CuI (0.3 equiv) were used (>99:1 anti/syn). They reasoned that the high selectivity arose from the use of a monodentate Lewis acid and the highly dissociated anion derived from the organocopper species.Fujisawa studied the addition of lithiated dithiane to (S)-1  66]. Wi. WiS)-1 S)-1 , no anti-adduct 29 was detected, instead the syn-adduct 30 was obtained as the sole stereoisomer. By lowering the temperature to −40 °C and keeping the amount of reagents the same, the selectivity was inverted! Only the anti-adduct 29 was isolated. This change in selectivity was explained with two different transition states. At lower temperatures (−40 °C) under kinetic control the Felkin–Anh product is predominant. At higher temperatures (−15 °C) the nucleophilic addition occurs via a thermodynamically more stable transition state that resembles the chelation control TS. These results are interesting as this is the first time anyone reports a complete inversion of selectivity just by raising the reaction temperature. Decrease in anti-selectivity has been evidenced by others, when the reaction temperature has been raised by 40 to 100 °C , but never a total reverse. If these results are reliable, there has to be a total change in the transition state towards total chelation control and most likely also in the aggregation level of lithiated reagents.Recently Lam reported interesting results on the stereoselective addition of a lithiated alkynyl species to (S)-1  67]. Ac. AcS)-1 31 was used as the nucleophile, their results were similar to Herold’s . Th. Thα-prosyn-selectivities. With propyneCuI the selectivity was 1:16 (anti/syn) providing the propargylic alcohol 43 in 95% yield. By changing the nucleophile to TMS-ethyneCuI the syn-selectivity increased to over 1:20, but providing the syn-adduct 44 in lower yield (82%). HPLC analysis showed that no epimerization had occurred. These results support Herold’s seminal work published 20 years earlier . In. In72].  earlier . Reginat control . Surprisreaction . After c in 1990 .. have examined the use of vinylzinc nucleophiles as alkenylating agents in the synthesis of D-erythro-sphingosine (S)-1 with pentadecenyl(ethyl)zinc (48) in the presence of catalytic (R)-diphenyl(1-methylpyrrolidin-2-yl)methanol -DPMPM) . Tr. Tr. hav)-DPMPM)  in tolue53 was performed with a trialkylsilane and a Ni(COD)2 catalyst ligated with an in situ formed N-heterocyclic carbene. In all cases studied, they found both the anti/syn- and the Z/E-selectivity to be high (>20:1), but the chemical yield was varying. Highest yields (78–80%) were achieved with short alkyl chains (R = Me) or when R = phenyl. When the alkyl chain was lengthened (R = C13H27), while aiming for the synthesis of D-erythro-sphingosine, the yield drastically dropped to 18%. By changing the reaction conditions (2 equiv of (S)-1 with 1% water in THF) the yield increased to acceptable levels (65%) while the selectivity remained practically the same. They also tested the reaction with other serinal derivatives, but the best results were achieved with (S)-1.Montgomery studied the nickel-catalyzed reductive additions of α-aminoaldehydes with silylalkynes  76]. Th. Th76]. E)-vinyl nucleophiles through hydrozirconation. In view of the relatively low electronegativity (1.2–1.4) of Zr, which is roughly comparable with that of Mg and somewhat lower than that of Al, the low reactivity of organylzirconocene chlorides towards carbonyl compounds is puzzling. It is likely that the presence of two sterically demanding cyclopentadienyl (Cp) groups is at least partially responsible for their low reactivity. Suzuki noticed that Lewis acids promote C–C-bond forming reactions of organylzirconocene nucleophiles , but no diastereoselectivity was observed. Peter Wipf has done pioneering work on the hydrozirconation–transmetallation sequence . Th. ThE)-visequence –79. Murath (S)-1 –47. When31 as the nucleophile, which had been investigated by Negishi for the hydrozirconation–transmetallation process . We. We48].  process . Our findduct 59 . Poor yiZ)-vinyl nucleophiles created from the vinyl iodide 61 in the stereoselective synthesis of pachastrissamine (11) . T. T64 thald 27%) . With a ium salt . These c% . With Z-selectivity is a typical outcome with unstabilized ylides. Kim has investigated means of reversing the E/Z-selectivity to favour the E-isomer . As. As1 witylide 69 . They obZ-enoate 73 [Z-enoates possible [3CH2O-groups destabilize the cis-oxaphosphetane intermediate . HPLC analysis showed that there was no epimerization.We have been interested in the synthesis of noate 73 . The Stipossible . The twormediate  and make74 reacts with the Still–Gennari phosphonate and provides the Z-enoate 75 in good E/Z-selectivity (1:12) [E/Z-selectivity can be reasoned with the smaller size of the aldehyde 74. When the aldehyde is coordinating to the phosphonate, the steric hindrance caused by the interaction of the trifluoroethoxy group with the aldehyde R’ in TS (syn) is smaller compared with aldehyde 1. This allows some of the aldehyde to react via the trans-oxaphosphetane intermediate. Using these reaction conditions no epimerization was observed.We have recently shown that the open-chain aldehyde y (1:12) . The sliGarner’s aldehyde has developed into a useful and reliable synthetic intermediate for the synthesis of enantiopure complex natural products, their analogues and other pharmacologically active compounds. Several reaction types have been studied sufficiently so that one has reliable tools to plan a synthesis. Simple addition reactions to the carbonyl group give access to vicinal amino alcohols, important building blocks for many natural products. Another possibility for carbon chain elongation are olefination reactions, which often lead to epimerization of the α-stereocenter. As with the alkylations, strategies to avoid this undesired reaction have been developed. However, despite these important achievements, much more research needs to be done to increase the scopes of these reactions, the overall efficiency, and environmental sustainability.


Enterococcus faecalis, a better understanding of the molecular mechanisms, particularly of antibiotic detection, signal transduction and gene regulation is needed. Because molecular studies in this bacterium can be challenging, we aimed at exploiting the genetically highly tractable Gram-positive model organism Bacillus subtilis as a heterologous host. Two fundamentally different regulators of E. faecalis resistance against cell wall antibiotics, the bacitracin sensor BcrR and the vancomycin-sensing two-component system VanSB-VanRB, were produced in B. subtilis and their functions were monitored using target promoters fused to reporter genes (lacZ and luxABCDE). The bacitracin resistance system BcrR-BcrAB of E. faecalis was fully functional in B. subtilis, both regarding regulation of bcrAB expression and resistance mediated by the transporter BcrAB. Removal of intrinsic bacitracin resistance of B. subtilis increased the sensitivity of the system. The lacZ and luxABCDE reporters were found to both offer sensitive detection of promoter induction on solid media, which is useful for screening of large mutant libraries. The VanSB-VanRB system displayed a gradual dose-response behaviour to vancomycin, but only when produced at low levels in the cell. Taken together, our data show that B. subtilis is a well-suited host for the molecular characterization of regulatory systems controlling resistance against cell wall active compounds in E. faecalis. Importantly, B. subtilis facilitates the careful adjustment of expression levels and genetic background required for full functionality of the introduced regulators.To combat antibiotic resistance of  Enterococcus faecalis is one of the most common causes of nosocomial infections. Increasing incidences of infections with antibiotic resistant strains, particularly with vancomycin resistant enterococci (VREs), therefore pose a major health risk E. faecalis in its natural gut habitat. Furthermore, many enterococcal isolates were found to be highly resistant against bacitracin BBXB proteins mediate resistance E. faecalis is conferred by the ATP-binding cassette (ABC) transporter BcrAB, which presumably removes the antibiotic from its site of action (i.e. the cytoplasmic membrane) The molecular mechanisms leading to resistance are often well known. In the case of vancomycin, high-level resistance is for example ensured by target alteration through replacement of the terminal D-Ala-D-Ala by D-Ala-D-lactate. In VanA-type strains, this is accomplished through the action of the VanHAX system, while in VanB-type strains the VanHvan operons are induced in the presence of vancomycin by the two-component systems VanS-VanR or VanSB-VanRB for VanA- and VanB-type resistance, respectively bcrAB is mediated by the one-component transmembrane regulator BcrR B respond to vancomycin, their sensory domains differ considerably in size with 37 amino acids for VanS and 103 residues for VanSB, and share only low sequence similarity The expression of most resistance genes is induced in the presence of the respective antibiotic. For example, the E. coliE. faecalisBacillus subtilis has been used successfully for the functional expression of the VanS-VanR two-component system of E. faecalis, as well as of the VanB-type resistance proteins E. coli, B. subtilis is easy to manipulate and a large number of genetic tools are available. The G+C contents of B. subtilis (43.5%) and of E. faecalis (37.5%) are comparable, which is of great advantage for heterologous gene expression. Furthermore, the transcription machinery in both organisms is sufficiently similar to facilitate the interaction of heterologous transcriptional regulators with the native machinery, as has been shown in vitro for activation of B. subtilis RNA polymerase by E. faecalis BcrR B. subtilis against cell wall antibiotics are well understood Sensory perception of antimicrobial substances by bacteria is a first and essential step in antibiotic resistance, and a thorough understanding of the mechanisms involved would provide an important basis for the development of new drugs to combat resistance. However, in many genera, e.g. the enterococci, investigations are hampered by the difficulty to manipulate these bacteria genetically. Although more and more genetic tools are becoming available for enterococci, poor transformability of many strains, including clinical isolates, still impedes studies involving, for example, high-throughput or detailed mutagenic approaches. To circumvent these problems, heterologous hosts have been chosen, often using E. faecalis to develop and validate B. subtilis as a platform for studying the regulatory mechanisms leading to resistance against cell wall-active antibiotics. To test the feasibility of our approach and determine the optimal genetic background of the host, we chose the one-component regulator BcrR and could show full functionality with highly similar behaviour to its native context. This set-up was then applied to the VanSB-VanRB two-component system. A previous attempt at heterologous expression of this system in B. subtilis had resulted in a constitutively active behaviour B. subtilis as host organism.In the present study, we have used two well-understood examples from E. coli DH5αand XL1-blue were used for cloning. E. coli and B. subtilis were grown routinely in Luria-Bertani (LB) medium at 37°C with agitation (200 rpm). B. subtilis was transformed by natural competence as previously described −1 for E. coli), chloramphenicol (5 μg ml−1 for B. subtilis), kanamycin (10 μg ml−1 for B. subtilis), erythromycin 1 μg ml−1 with lincomycin 25 μg ml−1 (for macrolide-lincosamide-streptogramin B (mls) resistance in B. subtilis) or spectinomycin (100 μg ml−1 for B. subtilis). Bacitracin was supplied as the Zn2+-salt. Unless otherwise stated, media for strains carrying pXT-derived constructs contained 0.2% (w/v) xylose for target gene expression. Solid media contained 1.5% (w/v) agar. Growth was measured as optical density at 600 nm wavelength (OD600).All strains used in this study are listed in All primer sequences used for this study are listed in bcrA to lacZ or bacterial luciferase (luxABCDE) were constructed in vectors pAC6 vanYB to bacterial luciferase was cloned into the EcoRI and SpeI sites of vector pAH328 creating plasmid pCF133. The exact regions contained in the constructs are given in Transcriptional promoter fusions of PbcrR or bcrR-bcrAB in B. subtilis (pES701 and pES702) the respective DNA fragments were amplified from the plasmid pAMbcr1 xylA-promoter. Plasmid pCF132 was constructed by inserting BSBvanR from E. faecalis V583 into the BamHI and HindIII sites of vector pXT for heterologous, xylose-inducible expression in B. subtilis.For heterologous, xylose-inducible expression of B. subtilis were cloned into the vector pMAD Constructs for unmarked gene deletions in All constructs were checked for PCR-fidelity by sequencing, and all created strains were verified by PCR using appropriate primers.B. subtilis in MH medium were used as inoculum at a dilution of 1∶500. After 24 h incubation in the presence of two-fold serial dilutions of the antibiotic the MIC was scored as the lowest concentration where no growth was observed.All cultures were grown in Mueller-Hinton (MH) medium for antibiotic susceptibility assays 600 between 0.4 and 0.5. The cultures were split into 2 mL aliquots and challenged with different concentrations of bacitracin with one aliquot left untreated. After incubation for an additional 30 min at 37°C with aeration, the cultures were harvested and the cell pellets were frozen at −20°C. β-galactosidase activities were determined as described, with normalization to cell density Cells were inoculated from fresh overnight cultures and grown in LB medium at 37°C with aeration until they reached an ODB. subtilis strains were assayed using a Synergy 2 multi-mode microplate reader from BioTek controlled by the software Gen5. LB medium was inoculated 1∶500 from overnight cultures, and each strain was grown in 100 μl volumes in a 96-well plate. Cultures were incubated at 37°C with shaking (intensity: medium), and the OD600 was monitored every 10 min. At an OD600 of 0.02 , either bacitracin was added to final concentrations of 0.03, 0.1, 0.3, 1 μg ml−1, or vancomycin to final concentrations of 0.01, 0.025, 0.05, 0.25 μg ml−1; in all cases one well was left untreated. Cultures were further incubated for 2 h, and the OD600 and luminescence  were monitored every 5 min. OD600 values were corrected using wells containing 100 μl LB medium as blanks. Raw luminescence output  was normalized to cell density by dividing each data-point by its corresponding corrected OD600 value (RLU/OD).Luciferase activities of E. faecalis, expression of the genes bcrAB that encode the bacitracin resistance transporter BcrAB is controlled solely by the one-component regulator BcrR B. subtilis, we introduced a transcriptional fusion of its target promoter, PbcrA, to lacZ (pES601), together with an expression construct of bcrR controlled by a xylose-inducible promoter (pES701), into the wild-type strain. Addition of increasing concentrations of bacitracin led to a strong upregulation (approximately 80-fold) of promoter activities with a threshold concentration for induction of 0.3 μg ml−1 . Here, the threshold for induction was ten-fold lower at 0.03 μg ml−1 bacitracin, with 0.1 μg ml−1 leading to full induction. Furthermore, the maximal amplitude of induction was significantly increased (p = 0.006) to more than 200-fold  into TMB035 (bceAB::kan). In this strain, the induction behaviour was comparable to that of wild-type B. subtilis carrying BcrR alone  of bacitracin was strongly reduced from 128 μg ml−1 in the wild-type to 2–4 μg ml−1 in the bceAB-deleted strain TMB035 (bcrR-bcrAB construct increased the resistance of the bceAB-deleted strain to 32 μg ml−1 (B. subtilis (i.e. 8- to 16-fold increase in MIC) is the same as that conferred to E. faecalis itself, where BcrAB raises the MIC from 32 μg ml−1 to >256 μg ml−1E. faecalis transporter in wild-type B. subtilis could not further increase its resistance  per OD) was observed in the absence of bacitracin or in a strain lacking BcrR , consistent with the low promoter activities reported above for this strain. The reporter strain harbouring BcrR and the PbcrA-lux construct showed strong luminescence when grown on agar plates containing 0.3 μg ml−1 bacitracin, and no detectable luminescence in its absence  DNA-libraries, assays performed on solid media greatly facilitate screening of large numbers of clones. To evaluate the  absence . As obse absence .E. faecalis, where it is much more difficult to obtain large numbers of transformants than in the naturally competent B. subtilis. Importantly, the output of both promoters is sufficiently sensitive to allow assays to be performed at sub-lethal concentrations of the antibiotic, at least in the case of the Bcr-system. The feasibility of this approach was recently demonstrated in a study that identified essential residues in the B. subtilis bacitracin resistance transporter BceAB Both reporter constructs are therefore suitable for screening libraries of clones for promoter induction and are applicable for high-throughput approaches. In principle, screens for loss-of-function as well as gain-of-function mutations can be performed, depending on experimental design. This set-up offers a great advantage over studies performed directly in E. faecalis to B. subtilis, we next wanted to test if our set-up could be applied to other regulatory systems. The two-component system VanS-VanR regulating VanA-type vancomycin resistance had previously been shown to be functional in B. subtilisBvanSBvanR encoding the regulatory system for VanB-type resistance had resulted in constitutive expression of the target promoter, PvanYB, and the authors could show that this was due to constitutive activity of the sensor kinase VanSB under the conditions chosen B activity could be obtained by optimization of conditions, we introduced an expression construct of the BvanSBvanR operon under control of the xylose-inducible promoter PxylA into TMB1518. The activity of the two-component system was monitored as activation of a PvanYB-luxABCDE transcriptional fusion. In the absence of xylose, only low levels of the two-component systems will be produced in the cell, due to basal promoter activity of PxylA. Under these conditions, addition of increasing concentrations of vancomycin to growing cultures of the reporter strain led to a gradual up-regulation of promoter activity  screening approaches, for example of mutant libraries, as well as for the quantitative characterization of regulators. Complementation studies with random or directed mutations can thus be initiated in the genetically accessible, highly competent host B. subtilis, and promising results then validated directly in E. faealis. Construction of the desired heterologous strains will be further aided by a recently established and fully validated tool-box of vectors, promoters, reporters and epitope-tags for engineering of B. subtilisFurther, we showed that the two reporters,


Using word-counting methods, we indexed the existence of every 8-mer oligo composed of bases A, C, G, T, r, y, s, w, m, k, n or 12-mer oligo composed of A, C, G, T, n, in the promoters of all predicted genes of Arabidopsis thaliana genome and of selected stress-induced co-expressed genes. From this analysis, we identified number of over-represented motifs. Among these, major critical motifs were identified using a position filter. We used a model based on uniform distribution and the z-scores derived from this model to describe position bias. Interestingly, many motifs showed position bias towards the transcription start site. We extended this model to show biased distribution of motifs in the genomes of both A. thaliana and rice. We also used MotifIndexer to identify conserved motifs in co-expressed gene groups from two Arabidopsis species, A. thaliana and A. lyrata. This new comparative genomics method does not depend on alignments of homologous gene promoter sequences.The discovery of DNA regulatory motifs in the sequenced genomes using computational methods remains challenging. Here, we present MotifIndexer - a comprehensive strategy for  The mechanisms behind regulated gene expression have been subject of passionate discussions for generations of molecular biologists. Though the list for such mechanisms has constantly been broadened, promoter motif based regulation remains an extensively studied topic of importance for eukaryotic gene expression. DNA regulatory motifs, or cis-regulatory elements, are 5–15 base pair (bp) long nucleotides within the promoter region that function primarily as transcription factor (TF) binding sites, although binding for other proteins, such as kinases, has also been reported It is assumed that co-expressed genes are likely to contain a set of over-represented motifs in their promoters that lead to their similar expression patterns. Various computational methods have been developed to discover such motifs. These methods can be divided into two categories based on their ways to represent motifs: those using a probabilistic sequence model and those using word-based methods Drosophila, and mammals bona fide motifs.As more complete genome sequences are available, comparative genomics can be used for motif discovery. These methods aim to identify evolutionarily conserved non-coding sequences via sequence alignment, usually within the promoter region of homologous gene sets from related species. Such a strategy has been used to discover motifs from yeast, A. thaliana genes and of selected stress-induced co-expressed genes Here, we describe an algorithm termed MotifIndexer that is based on word counting strategy. We have successfully used this algorithm to identify motifs from promoters of all predicted 8 oligos in this format cover almost all versatility in terms of nucleotide degeneration. Only wobble bases representing three different nucleotides will be missed in this format. The second format extracts 12-mer oligos, with all possible combination of A, C, G, T, or n. This format, with limited complexity in degeneration, allows characterizing motifs up to 12 bp, and the total number of oligos is 244,140,625 or 512.In word-counting based motif discovery algorithms, the format used to represent motifs is usually limited by search space volume. To balance between motif length and motif complexity, we chose two different motif formats. The first considers 8-mer oligos, with all possible combination of A, C, G, T, r(A|G), y(C|T), s(C|G), w(A|T), m(A|C), k(G|T), or n(A|C|G|T). The 214,358,881 or 11A. thaliana genome, or within any selected group of promoters. The procedure for cataloging the 8-mer oligos is shown in For every 8- or 12-mer oligo, we developed an algorithm to efficiently catalog the number of promoters harboring it within the Once oligo numbers are cataloged for the whole genome and for promoters of a specific group of co-expressed genes, a pValue is calculated for every 8-mer or 12-mer oligo based on the hypergeometric distribution. This pValue provides a measure for over or under-representation of the corresponding oligo in the selected groups of promoters. Our method guarantees the finding of all 8-mer oligos with pValue smaller than a selected cut-off value. In the following analysis, we focused on oligos existing in fewer than 50% of all the promoters in the genome, which include 75,029,949 8-mer oligos and 242,575,400 12-mer oligos.To test our algorithm, we selected co-expressed gene groups consisting of 7,424 genes from a previous study that mainly includes Arabidopsis genes that are differentially regulated during biotic and abiotic stress responses All 93,138 over-represented 8-mer oligos with pValues < = 1E-05 for cluster_N0 are shown in The over-represented oligos can also be sorted by their fold enrichment in the group of selected promoters relative to the whole genome. For example, in cluster_N0, among the oligos with similarities to the major motifs, many showed higher fold change enrichment than the major motifs themselves. bona fide motifs in many studies Typically, promoter motifs show position bias towards the transcription start sites (TSS), a feature that has been used as supporting evidence for 2−1)/n. Thus, for any 8-mer oligo with n instances within any selected promoters, a z-score is calculated as:While the distribution plot depicted in where p is the average position location. For orientation, we describe p = 1 as the position at −1000 relative to TSS, while p = 993 is the position immediately adjacent to TSS.Evenly distributed oligos should have a z-score close to 0. A large positive z-score indicates the oligo has a higher occurrence in the region between 0 to −500 relative to the TSS than in the −500 to −1000 region. That is, it has its positions biased towards the TSS. The mean position and z-score for the top motif from most clusters are listed in In our analysis, we also observed a correlation between small pValues and positive z-scores for co-expressed stress responsive genes. Such correlations for genes in cluster_N0 are illustrated in bona fide motifs.To evaluate the false discovery rate (FDR) in our algorithm, we repeated the analysis on 100 groups of randomly selected promoters, with 50 promoters in each group. The number of oligos resulting from these analyses should be similar to the number of false positive oligos identified in an actual experiment. On average, each group had 1.76, 17.8, or 180 oligos (before position-based filter) with pValue smaller or equal to 1E-7, 1E-6, and 1E-5, respectively. These numbers are close to the expected value: 75,029,949 X pValue/4. Among the 180 oligos with pValue < = 1E-5, only 0.49 oligo has its TSS z-score larger than 3. Analysis on groups of promoters with size 200 or 500 returns slightly (∼10%) more oligos that we would consider to be false positive. Based on these results, we choose the cutoff values in our analysis to minimize the number of false discovered oligos while maximizing the number of We used two criteria for qualifying an oligo to be a potential motif. The first one is based on the pValue cut off, and the second one is based on the z-score cut off. For 8-mer oligos, we took all those with pValue <1E-7, and those with pValue between 1E-5 and 1E-7 and z-score >3 as potential motifs. It should be noted that, though the pValue and z-score show correlation, these are two different measurements and are independent of each other. Therefore, motifs qualifying for both criteria should have high confidence to be a true motif. Using these criteria, we identified all 8-mer motifs that are over-represented in various co-expressed gene clusters . Among tGenes in cluster_N19 are induced by pathogen elicitors/effectors or PAMPs and have roles in defense. As expected, two W-Box like motifs “TTGACTTy" and “kGTCAAm" are highly over-represented in this cluster of co-expressed genes . WRKY tr2+/calmodulin-binding transcription factor AtSR1  has been shown to bind to the CGCG motif within the promoter of the EDS1 gene acting as a repressor of its expression Genes in cluster_N0 are induced by biotic and abiotic stresses. The over-represented motifs “rGTCAAm" and “AAAGTCww"  are relaGenes in cluster_N12 are induced by a variety of abiotic or biotic stresses. These genes are often viewed as common stress responsive genes. In this cluster, motif “mCGCGT" and the related motifs “ACrCGy" and “rCrCGkmm" are highly represented , all thrPromoters of genes from several other clusters used in this study are induced by the plant stress hormone abscisic acid (ABA) at early time points (N3) or late time points , and also by other abiotic stresses. As reported earlier Our analysis also identified many motifs from N18, N14, N2, and N5 cluster genes that are down-regulated during stress . ClusterIt has been reported that transcription factor binding motifs from eukaryotes are 6–10 bp long. Our 12-mer motif analysis supports this notion. By including letter “n" in the motifs, our algorithm revealed that the length of most core motifs is less than 9 bp . For exaOne exception is found among motifs identified from cluster_N7. Genes in this cluster are down-regulated by stresses and mainly expressed in xylem tissue of roots. The motif “CGTGnGnGGCAC" is over-represented in this cluster, with highly biased positioning towards TSS.Collectively from this analysis, we conclude that the analysis on 8-mer oligos captures most motifs, while the analysis on 12-mer complements and enforces the information gained from 8-mer oligo analysis and may recover a few additional long motifs.Pseudomonas syringae pv. tomato (Pst) expressing avrRpm1 effector. This analysis identified 461 genes that were up-regulated more than two-fold in Pst expressing avrRPM1 after 6 hours compared to the mock control . Our analysis of the promoters of these genes using MotifIndexer algorithm are listed in We tested if our algorithm could be used to identify over-represented motifs from dataset that are generated from single microarray experiments. The data were extracted from AtGenExpress and include Arabidopsis responses to the bacterial pathogen A. lyrata is a close relative of A. thaliana. The coding regions between the two genomes share ∼92% identity, while the promoter regions have ∼85% identity A. thaliana, we chose those have orthologous promoters in A. lyrata and subjected them to MotifIndexer analysis. The same procedure was carried out for the orthologous promoters in A. lyrata. For each 8-mer oligo, pValues from each species were obtained and the larger value of the two was assigned as a conserved pValue to that 8-mer oligo.A. thaliana, while only 12 of them were conserved among the two species. Thus, 99% of random noises or false positive oligos can be removed by comparing the two genomes. And less than 1 oligo (0.45 in average) was left if the conserved pValue was set at pValue < = 1E-05. Similar results were obtained for 100 groups of 100 randomly chosen promoters. On the other hand, for promoters from cluster_N0, there were 164,215 oligos with pValue < = 1E-04 in A. thaliana, and 75,371 of them also have associated pValue < = 1E-04 in A. lyrata. Thus, 46% of the oligos are conserved among the two species. Similar results were also obtained for promoters in other clusters (data not shown). The conserved motifs in cluster_N0 identified via this procedure are listed in A. thaliana alone. The other four motifs demonstrate the power of such comparative genome analysis. They share similar pValues and position bias distribution in both species, with conserved pValue between 1E-05 and 1E-08. Comparing with single species analysis, their pValues did not change, but the corresponding cut-off pValue was 2 orders larger. Thus, these motifs that would have been considered less significant in a single species analysis were more significant in a comparative genome analysis. This indicates comparative genome analysis using MotifIndexer can identify novel motifs.The analysis was performed on groups of randomly chosen promoters, or on groups of promoters from co-expressed genes. For 100 groups of 30 randomly chosen promoters, there were on average 1,730 oligos with pValue < = 1E-04 in We compared results obtained from MotifIndexer with two word-based motif finding software Weeder and Amadeus The search for motifs with position bias towards TSS can also be applied to all promoters in a genome. To this end, we applied our algorithm to both the Arabidopsis and the Rice genomes. For Arabidopsis, we included the promoters from 17,461 genes that have a 5′ untranslated regions (UTR) of at least 50 bp in length. The promoters chosen extended 1000 bp upstream of the 5′ UTR. For rice, we included 22,493 genes’ promoters with the same criteria.For both Arabidopsis and rice, we calculated the z-score for 8-mer motifs with less than or equal to 2 base pairs as degenerative, with the degenerative bases limited to r, y, s, w, or n. This analysis resulted in 28 oligos with obvious position bias in both the Arabidopsis and the rice genome . Of thesIn addition to identifying motifs that show strong position bias in both Arabidopsis and rice, we identified motifs with position bias in only one species, either Arabidopsis  or rice Here, we describe an algorithm that identifies all over-represented 8-mer oligo in groups of promoters that meet a predetermined pValue cut off, except for those oligos with wobble bases representing three nucleotides. It should be noted that wobble bases representing three nucleotides are rarely used for motif discovery A. lyrata is a close relative of A. thaliana. The homologous promoters from these two species share similarity levels between 75% and 95%. Under these similarity levels, the true motifs are expected to be conserved between the two species, while the false positive oligos are not. Since MotifIndexer calculated a pValue for every possible 8-mer motif, it can be easily adapted for comparative genomic analysis, as shown for cluster_N0 promoters . The dataset contains the upstream 1000 base pairs for 33,518 genes. The rice upstream sequences were downloaded from the Rice Annotation Project Database (http://rapdb.dna.affrc.go.jp/). The IRGSP/RAP build 5 dataset was used to extract the 1000 base pair sequences upstream of 5′UTR from those genes with 5′UTR longer than 50 bp.Arabidopsis promoter dataset was downloaded from TAIR (http://dinesh-kumarlab.genomecenter.ucdavis.edu/downloads.html) and upon request for academic use.The procedure to catalog the number of promoters containing specific 8-mer or 12-mer oligos is illustrated in M promoters in total, an oligo presents in m promoters among them. And within the K promoters in the whole genome, the oligo presents in k promoters. If m is larger than the expected value, that is m > k *M/K, a pValue is calculated as:a is the largest integer between 0 and k *M/K that fulfills the requirement:For pValue calculation of oligos, we only consider those presented in less than half of genome’s promoters, which includes 75029949 8-mer oligos and 242575400 12-mer oligos. Suppose in a group of selected genes with After pValues were calculated for all the over-represented oligos, they were sorted by their pValues. And for any pair of reverse complemented oligos, only one oligo is retained. Then, the selected promoters were scanned for the presence of the oligos to calculate their average position and z-score for TSS. Finally, major motifs were picked as described in the result section.The process was carried out on selected co-expressed gene clusters, as well as randomly selected promoter groups.A. lyrata was downloaded from http://genome.jgi-psf.org/Araly1/Araly1.download.ftp.html. The protein sequences of A. lyrata (Filtered Models6) were blasted against the A. thaliana protein sequences, and homologous gene pairs were selected based on these criteria: a, having at least 80% identities in amino acid sequences; b, the length of the A. lyrata protein is between 0.8 to 1.2 fold of that of the A. thaliana; c, potential duplicated genes in A. lyrata were filtered out. In total, 19,938 genes from A. lyrata were left and their promoters were extracted as 1,000 base pairs up stream of the translation start codon, and the same was done for A. thaliana. The analysis was carried out as described in the result section.The genome sequence of Table S1Top motifs have position bias.(DOC)Click here for additional data file.Table S2Major 8-mer motifs identified for coexpressed genes from various clusters.(DOC)Click here for additional data file.Table S3Major 12-mer motifs over-represented in stress responsive genes promoters.(DOC)Click here for additional data file.Table S4Major motifs identified from Arabidopsis genes induced by Pseudomonas syringae pv. tomato (Pst) (avrRpm1) – a case study on a single microarray experiment.(DOC)Click here for additional data file.Table S5Motifs with position bias only in Arabidopsis.(DOC)Click here for additional data file.Table S6Motifs with position bias only in Rice.(DOC)Click here for additional data file.